- Email: [email protected]
Molecular characterisation of Miamiensis avidus (Ciliophora: Scuticociliata) from ranched Southern bluefin tuna, Thunnus maccoyii off Port Lincoln, South Australia
Accepted Manuscript Molecular characterisation of Miamiensis avidus (Ciliophora: Scuticociliata) from ranched Southern Bluefin tuna, Thunnus maccoyii off Port Lincoln, South Australia
Jimena Balli Garza, Nathan J. Bott, Michael D. Hammond, Natalie Shepherd, Barbara F. Nowak PII: DOI: Reference:
S0044-8486(16)30497-5 doi: 10.1016/j.aquaculture.2016.11.040 AQUA 632437
To appear in:
aquaculture
Received date: Revised date: Accepted date:
17 September 2016 21 November 2016 29 November 2016
Please cite this article as: Jimena Balli Garza, Nathan J. Bott, Michael D. Hammond, Natalie Shepherd, Barbara F. Nowak , Molecular characterisation of Miamiensis avidus (Ciliophora: Scuticociliata) from ranched Southern Bluefin tuna, Thunnus maccoyii off Port Lincoln, South Australia. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Aqua(2016), doi: 10.1016/ j.aquaculture.2016.11.040
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1. Institute for Marine and Antarctic Studies, University of Tasmania, Locked Bag 1370, Launceston 7250, Tasmania, Australia 2. Centre for Environmental Sustainability and Remediation, School of Science, RMIT University, PO Box 71, Bundoora 3083, Victoria, Australia.
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*-Corresponding author: Nathan Bott, Centre for Environmental Sustainability and Remediation, School of Science, RMIT University, PO Box 71, Bundoora 3083, Victoria, Australia. Ph. +61 3 99257143, Fax +61 3 99257110, Email. [email protected]
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† equal first authors
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Abstract Scuticociliates are opportunistic protozoan pathogens present in a wide range of teleost hosts. Uronema spp. and Miamiensis spp. are the two most common genera recorded from scuticociliatosis cases in farmed and ornamental fish. Southern bluefin tuna (Thunnus maccoyii) (SBT) ranching is a high value aquaculture sector, situated off Port Lincoln, South Australia. Uronema nigricans has been previously associated with SBT swimmer mortality syndrome and was considered to be the causative agent. We conducted the first molecular characterisation of swimmer syndrome agent from affected SBT. Comparison of SSU rDNA and mitochondrial cytochrome c oxidase 1 sequences from the cerebrospinal fluid from SBT affected by swimmer syndrome and Uronema marinum samples, and phylogenetic analyses identified the scuticociliate present in SBT samples as Miamiensis avidus. Bayesian Inference analyses of both partial gene sequences of the Port Lincoln isolates form a clade with known M. avidus to the exclusion of Uronema spp. This shows that M. avidus is associated with swimmer syndrome and is present in the environment around SBT leases. Based on our molecular data, there is no evidence of Uronema spp. presence in the infected SBT. This is the first time M. avidus has been documented in Australia.
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Jimena Balli Garza1†, Nathan J. Bott2*†, Michael D. Hammond2, Natalie Shepherd2, Barbara F. Nowak1
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Molecular characterisation of Miamiensis avidus (Ciliophora: Scuticociliata) from ranched Southern Bluefin tuna, Thunnus maccoyii off Port Lincoln, South Australia
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Key words: Miamiensis avidus, Thunnus maccoyi, Scuticociliates, molecular characterisation, Southern bluefin tuna
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Introduction Scuticociliates are free-living marine organisms which feed on suspended bacteria, microalgae or other protozoa. Under certain circumstances scuticociliates
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can behave as opportunistic histophagous parasites of marine fish (Elston et al.,
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1999; Lee et al., 2004; Moustafa et al., 2010). They are considered to be serious
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pathogens in fish mariculture (Budiño et al., 2011; Gao et al., 2012, 2010; Whang
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et al., 2011) and were responsible for causing mortalities in olive flounder
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Paralichthys olivaceus (see Iglesias et al., 2001; Jung et al., 2005; Moustafa et al.,
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2010), turbot Scophthalmus maximus (see Dyková and Figueras, 1994; Iglesias et
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al., 2001; Sterud et al., 2000; Whang et al., 2013), sea bass Dicentrarchus labrax
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(see Whang et al., 2013), Southern bluefin tuna Thunnus maccoyii (SBT) (Munday
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et al., 1997, 2003), New Zealand grouper Polyprion oxygeneios and Yellowtail
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kingfish Seriola lalandi (Smith et al., 2009).
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The SBT industry is a high value aquaculture sector situated off Port
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Lincoln, South Australia. The industry practices purse seine fishing to collect 2-
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4year age class SBT in the Great Australian Bight, towing the fish back to the
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commercial lease sites and fattening over a 6 month period. Marine scuticociliates
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are the causative agent of swimmer syndrome in SBT, the disease typically occurs
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between May and November when water temperature drops below 18°C and, in
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most cases is associated with water temperature below 15°C (Deveney et al.,
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2005; Nowak et al., 2007). Clinical signs include abnormal and vigorous swimming
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at the surface followed by death (Munday et al., 1997). Affected fish are
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characterised by significant pathological changes in the olfactory rosettes and brain
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(encephalitis) along with the presence of scuticociliates. These ciliates have been
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identified as Uronema nigricans based on morphological analysis of cultures from
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cerebrospinal fluid (CSF) of infected SBT (Munday et al., 2003).
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In 2003, 58% of SBT mortalities examined from a mortality outbreak in
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winter were identified as being positive for scuticociliates based on the presence of
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scuticociliates in CSF (Deveney et al., 2005). This outbreak was affecting SBT
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from cages belonging to one company. Improved husbandry and feeding practices
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in the industry have decreased disease prevalence, however sporadic outbreaks
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still occur (Deveney et al., 2005; Nowak et al., 2007; Nowak, 2007).
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The use of morphological features to identify ciliates can be difficult (Jung et al., 2007; Song et al., 2009). Scuticociliates display considerable morphological
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plasticity, especially when cultured in vitro (Budiño et al., 2011; Salinas et al.,
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2012). Molecular techniques can be used to identify scuticociliates (Whang et al.,
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2013). Analysis of ribosomal RNA (rRNA) subunits has proven useful for
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identification and comparison of closely related organisms, including congeners
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(Elwood et al., 1985; Hillis and Dixon, 1991), while the application of mitochondrial
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barcoding of cytochrome c oxidase 1 (cox1) gene has been increasingly used for
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species delineation (Hebert et al., 2003; Robba et al., 2006; Roe and Sperling,
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2007). The use of sequencing data of rRNA genes and cox1 is a universally
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applicable tool that makes it possible to identify scuticociliates and confirm
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taxonomic relationships previously established on the basis of ultrastructural and
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other morphological characteristics (Budiño et al., 2011; Elwood et al., 1985; Hillis
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and Dixon, 1991; Jung et al., 2005; Whang et al., 2013).
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In this study we document the molecular characterisation of scuticociliates isolated from CSF of suspected swimmer syndrome SBT mortalities and record the
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first identification of the scuticociliate Miamiensis avidus (senior synonym of
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Philasterides dicentrachi (Budiño et al., 2011; Jung et al., 2007)) from SBT (new
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host) in Australia (new location). We provide data that the scuticociliates isolated
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from SBT are distinct from the previously reported agent of swimmer syndrome
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Uronema nigricans.
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Materials and methods
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Field collection and processing of Southern Bluefin Tuna
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During 2015, 16 wild SBT caught from Pedra Branca off the Southern coast of Tasmania (45o51’00”S 146o58’12”E), were collected by long line fishing.
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Individual olfactory rosettes were removed from the 16 SBT. In addition, 23
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cerebrospinal fluid (CSF) samples (olfactory rosettes not available) were collected
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from ranched SBT which exhibited signs of swimmer syndrome, such as vigorous
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swimming inside the cage or at intervals swimming up to the surface and sinking
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again. SBT were opportunistically sampled and ranged between 15-40kg. CSF
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was sampled from fresh mortalities and moribund individuals using plastic Pasteur
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pipettes during the 2015 harvesting season from May to the second week of
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August, off Port Lincoln, South Australia (34o43’56”S 135o51’31”E). Prior to storage
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at -20oC, CSF subsamples were examined for the presence of live scuticociliates.
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Wet preparations of subsamples were examined and observed using a bright-field
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microscope at 100X and 400X and most were found to be positive. Samples were
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considered positive for scuticociliates if the microscopic observation revealed
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motile cells that were robust and granular in appearance (Jung et al., 2007) and
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pyriform shape (morphology consistent for scuticociliates). All samples (olfactory
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rosettes and CSF) were stored in nucleic acid preservation solution (4 M
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ammonium sulphate, 25 mM sodium citrate, 10 mM EDTA; pH 5.5) in a 1:5 ratio.
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All samples were stored on ice at approximately 4oC overnight and then at -20°C in
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the laboratory.
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Nucleic acid extraction and molecular analysis Total nucleic acid (TNA) was extracted from the RNA-later preserved olfactory rosettes and CSF samples using Bioline Isolate II Genomic DNA Kit
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(Bioline, Taunton, MA, USA), following the manufacturer’s instructions. TNA
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quantity of each sample was estimated using spectrophotometry (NanoDrop,
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Thermo Scientific, Waltham MA, USA). Additionally, DNA samples, courtesy of
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Prof. Iva Dyková (Brno University, Czech Republic), from cultured scuticociliates
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(Dyková et al., 2010) were provided; two of which were isolated in conjunction with
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Neoparamoeba perurans from fish hosts Psetta maxima (CESP/I) and Salmo salar
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(CTAS/I), and two from seaweeds Lithophyllum racemus (CLIT/I) and Palmeria
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palmata (CPAL2/1). These samples were also used for the molecular analysis and
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sequence comparison to scuticociliates from SBT. Partial fragments of the
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mitochondrial cytochrome c oxidase subunit 1 (cox1) gene (360 nucleotides) and
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the small subunit rDNA (SSU rDNA) (507 nucleotides) sequence were amplified
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using oligonucleotide primers previously reported by Jung et al., 2005 and Whang
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et al., (2013) (Table 1). PCR was performed in a final volume of 25 μl containing, 10 pM of each
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primer, 0.5 U of DNA Taq Polymerase (Bioline, Taunton, MA USA), 1.5 mM MgCl2,
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0.5 mM dNTPs, stabilizers and enhancers (MyTaq, Bioline, Taunton, MA USA),
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and DNA template. PCR amplification was performed in a Bio-Rad C1000 Touch
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Thermal Cycler (Bio-Rad, Hercules, CA USA). The following conditions were used
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to amplify partial SSU rDNA fragment, using primers Cil 2/Cil 4: at 95oC for 1 min
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(activation) and 40 cycles of 95oC for 15 s (denaturing), 50oC for 15 s (annealing),
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72oC for 15s (extension) and a final hold at 4oC. The PCR conditions used to
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amplify a partial SSU rDNA fragment using Cil 3/Cil 4 were 95oC for 1 min
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(activation) and 35 cycles of 95oC for 15 s (denaturing), 50oC for 15 s (annealing),
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72oC for 15s (extension) and 4oC as a final hold. The PCR conditions used to
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amplify a partial sequence of the cox1 gene of M. avidus using primers OX09-142,
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OX09-143 and a partial fragment of the cox1 gene of U. marinum using OX09-144,
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OX09-145 were: 95oC for 1 min (activation) and 30 cycles of 95oC for 15 s
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(denaturation), 50oC for 15 s (annealing) 72oC for 15 s (extension) and 4oC as a
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final hold.
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Amplified products were analysed in a 1.5% agarose gel by electrophoresis,
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stained with ethidium bromide and visualized using the UV transilluminator Gel Doc
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XR+ System (Bio-Rad, Hercules, CA USA). Amplified products were purified using
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the ISOLATE II PCR and Gel Purification kit (Bioline, Taunton, MA USA) and
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concentration was measured by spectrophotometry (NanoDrop, Thermo Scientific,
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Waltham MA, USA). Both strands, forward and reverse of the PCR products, were
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sequenced using ABI Big Dye Terminator v3.1 chemistry (Applied Biosystems,
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Foster City, CA USA) by the Australian Genome Reference Facility (AGRF),
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Parkville, Victoria.
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Alignment and Phylogenetic analysis Sequencher™ (GeneCodes Corp., Ann Arbor, Michigan, U.S.A, ver. 5.2.4) was used to produce consensus sequences from corresponding forward and
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reverse complemented sequences. Scuticociliate sequences from the appropriate
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gene regions (partial SSU rDNA and mt cox 1) were obtained via BLAST search
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(Altschul et al., 1990) (Tables 2 and 3) and aligned in conjunction with the
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sequences obtained as part of this study using the Clustal X (Thompson et al.,
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1997) accessory application in Bioedit® (Hall, 1999). Alignments were further
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refined by eye. Sequences produced as part of this study are listed by locality (Port
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Lincoln) and numbered based on their sample number. Bayesian Inference
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analyses of sequence alignments were conducted using; MrBayes® ver. 3.2.2
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(Ronquist and Huelsenbeck, 2003) using the parameters: ngen=2,000,000, nst =
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six, four Markov Chains used, burn-in was set to 100 and every 100th tree saved.
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The trees were based on a 50% majority rule consensus as per Aiken et al.,
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(2007). The trees produced from Bayesian analyses were viewed using Figtree ®
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(http://tree.bio.ed.ac.uk/software/figtree/). Multiple pairwise alignments were
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produced in Mega version 6 (Tamura et al. 2013) using number of differences.
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Partial SSU rDNA and mt Cox 1 sequences generated from this study were
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deposited in Genbank under the accession numbers KX842459-KX842468 (M.
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avidus SSU rDNA), KX842469-KX842477 (M. avidus mt Cox 1) and KX842478-
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KX842483 (U. marinum mt Cox 1).
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Results
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Molecular identification of scuticociliates Nineteen samples from CSF of ranched SBT were identified as positive for scuticociliates using PCR. None the olfactory rosettes samples from wild SBT
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considered negative. The scuticociliates isolated from CSF of SBT were 100%
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identical based on both partial SSU rDNA and mt cox1. Both partial SSU rDNA and
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mt cox1 showed that the scuticociliates isolated from SBT CSF were identical to
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published sequences of M. avidus and differed from Uronema spp. sequenced as
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part of this study and previously published sequences; partial SSU rDNA differed
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by 23 - 24 nucleotides and 95% similarity, and mt cox1 differs by a range of 34-35
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nucleotides and is 87.5-90% in range of similarity. Our sequencing results for SSU
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rDNA of U. marinum isolates from Dykova et al. (2010) showed that their
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sequences were 100% identical to those generated by Dyková et al. (2010) from
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the same samples. All U. marinum SSU rDNA sequences obtained in this study
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were 100% identical.
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Bayesian inference analyses of partial SSU rDNA (Figure 1) showed that the ten sequences from SBT ranched at Port Lincoln were 100% identical and
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homologous to published SSU rDNA sequences of M. avidus (Jung et al., 2011,
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2007; Paramá et al., 2006; Salinas et al., 2012). The SBT samples and M. avidus
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formed a clade with all other taxa (to the exclusion of the Uronema clade including
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those sequenced as part of this study (samples from Dyková et al., 2010)).
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The partial mt cox1 Bayesian Inference analysis (Figure 2) showed a similar
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topology to the SSU tree in that M. avidus formed a clade with Pseudocohnilembus
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spp. to the exclusion of the Uronema + Entodiscus borealis clade. Within the M.
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avidus + Pseudocohnilembus spp. clade, M. avidus formed a clade to the
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exclusion of Pseudocohnilembus spp.; the samples collected from Pt Lincoln as
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part of this study formed a clade to the exclusion of all other M. avidus.
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Discussion
The comparison of SSU rDNA and mt cox1 sequences of scuticociliates collected from SBT and U. marinum in association with comparison to published
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M. avidus was present and U. nigricans which was identified during earlier SBT
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ranching seasons as a cause of swimmer syndrome (Crosbie and Munday, 1997;
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Deveney et al., 2005; Munday et al., 1997, 2003), was absent. Previous
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identification was based on morphology of cultures of the ciliates from SBT CSF
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(Munday et al., 1997) and it has been shown that morphology of scuticocilliates
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can change in culture (Fenchel, 1990; Jee et al., 2001). Our phylogenetic analyses
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of 2 gene regions (SSU rDNA and mt cox1) (Figure 1 and 2) that are subjected to
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different evolutionary pressures (Gao et al., 2012; Jung et al., 2011a) clearly
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showed that the samples isolated from SBT swimmer syndrome samples were M.
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avidus, and based on these analyses they were not particularly close in sequence
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similarity to the Uronema sp. sequenced in this study.
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Our Bayesian Inference analyses support the work of Gao et al., (2012) in illustrating that the genera belonging to Philasterida: Miamiensis and Uronema are
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consistently positioned within different clades. Our study differed from Gao et al.,
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(2012) in that we have analysed only within the Philasterida while they compared
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SSU rDNA of a wide range of orders within the Scuticociliata. The partial SSU
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rDNA analysis positioned the Uronema clade to the exclusion of all other
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scuticociliates as opposed to that of the complete SSU rDNA in Gao et al., (2012);
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this may be an artefact of not analysing the complete gene region.
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While our results show that M. avidus is associated with swimmer syndrome
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in SBT further investigation is required to determine if Uronema nigricans (or
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indeed other opportunistic scuticociliate species) is also associated with swimmer
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syndrome in ranched SBT. If multiple species are causative agents and U.
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nigricans is also associated with swimmer syndrome in SBT (as described by
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Munday et al., 1997) then an investigation is warranted into whether environmental
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cues favour infection by one scuticociliate species over another. It is plausible that
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both scuticociliate species (M. avidus and U. nigricans) are responsible as they are
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Mixed isolates of scuticocilliates, including M.avidus, U. marinum and
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Parauronema virginianum were obtained from infected New Zealand grouper,
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Polyprion oxygeneios (see Salinas et al., 2011). Smith et al. (2009) isolated from
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dead P. oxygeneios and identified using SSU rRNA both M. avidus and U.
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marinum. However, in some host species only M. avidus has been suggested as
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the cause of scuticociliatosis. Only M. avidus induced histological changes when
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olive flounder Paralichthys olivaceus were experimentally infected with M. avidus,
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Pseudocohnilembus persalinus, P. hargisi and U. marinum. (Song et al., 2009).
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This suggested that P. persalinus, P. hargisi and U. marinum were not causative
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agents of scuticociliatiosis in olive flounder, although all three species have been
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reported and identified in the affected fish (Song et al 2009) and Smith et al. (2009)
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reported that U. marinum caused mortalities in Seriola lalandi in New Zealand.
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Culture conditions may promote growth of one species, and in some cases that
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species may not be the pathogen. For example, cultures of amoebae from the gills
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of Amoebic Gill Disease-affected Atlantic salmon resulted in isolation of N.
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pemaquidensis and N. branchiphila, while it was later shown that N. perurans is the
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only causative agent (Crosbie et al., 2012; Young et al., 2007). Further
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investigation is required to assess the broad pathogenicity of a range of
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scuticociliates believed to cause scuticociliatosis in finfish. This study is the first to
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document the presence of M. avidus in Australia. It is yet to be established how
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widespread M. avidus is in Australia’s marine environment.
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Acknowledgements
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The authors would like to thank the Australian Southern Bluefin Tuna Industry
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Association for their ongoing support, in particular Kirsten Rough and Claire
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Webber. We would also like to thank Dr Daryl Evans, Marnikol Fisheries for his
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ongoing support of SBT Health research. We are grateful to Prof. Iva Dyková for
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providing the DNA samples of Uronema spp. All work with animals and methods
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for recovering samples were approved by the University of Tasmania Animal Ethics
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Committee, project number A0013175. Samples from Czech Republic were
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imported under Quarantine permit IP15004906.
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Jung, S.J., Bae, M.J., Oh, M.J., Lee, J., 2011. Sequence conservation in the internal transcribed spacers and 5.8S ribosomal RNA of parasitic scuticociliates Miamiensis avidus (Ciliophora, Scuticociliatia). Parasitol. Int. 60, 216–219. doi:10.1016/j.parint.2011.02.004
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Jung, S.J., Kitamura, S.I., Song, J.Y., Joung, I.Y., Oh, M.J., 2005. Complete small subunit rRNA gene sequence of the scuticociliate Miamiensis avidus pathogenic to olive flounder Paralichthys olivaceus. Dis. Aquat. Organ. 64, 159–162. doi:10.3354/dao064159
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Jung, S.J., Kitamura, S.I., Song, J.Y., Oh, M.J., 2007. Miamiensis avidus (Ciliophora: Scuticociliatida) causes systemic infection of olive flounder Paralichthys olivaceus and is a senior synonym of Philasterides dicentrarchi. Dis. Aquat. Organ. 73, 227–234. doi:10.3354/dao073227
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Lee, E.H., Kim, S.M., Kwon, S.R., Kim, S.K., Nam, Y.K., Kim, K.H., 2004. Comparison of toxic effects of nitric oxide and peroxynitrite on Uronema marinum (Ciliata: Scuticociliatida). Dis. Aquat. Organ. 58, 255–260. Moustafa, E.M.M., Tange, N., Shimada, A., Morita, T., 2010. Experimental Scuticociliatosis in Japanese Flounder (Paralichthys olivaceus) Infected with Miamiensis avidus: Pathological Study on the Possible Neural Routes of Invasion and Dissemination of the Scuticociliate inside the Fish Body. J. Vet. Med. Sci. 72, 1557–1563. doi:10.1292/jvms.10-0214 Munday, B., O’Donoghue, P.., Watts, M., Rough, K., Hawkesford, T., 1997. Fatal encephalitis due to the scuticociliate Uronema nigricans in sea-caged, southern bluefin tuna Thunnus maccoyii. Dis. Aquat. Organ. 30, 17–25. Munday, B.L., Sawada, Y., Cribb, T., Hayward, C.J., 2003. Review Diseases of tunas, Thunnus spp. J. Fish Dis. 26, 187–206. Nowak, B., Aiken, H., Bott, N.J., Deveney, M., Johnston, C., McGowan, T.,
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Paramá, a, Arranz, J. a, Alvarez, M.F., Sanmartín, M.L., Leiro, J., 2006. Ultrastructure and phylogeny of Philasterides dicentrarchi (Ciliophora, Scuticociliatia) from farmed turbot in NW Spain. Parasitology 132, 555–64. doi:10.1017/S0031182005009534
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Robba, L., Russell, S.J., Barker, G.L., Brodie, J., 2006. Assessing the use of the mitochondrial cox1 marker for use in DNA barcoding of red algae (Rhodophyta). Am. J. Bot. 93, 1101–1108. doi:10.3732/ajb.93.8.1101
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Roe, A.D., Sperling, F.A.H., 2007. Patterns of evolution of mitochondrial cytochrome c oxidase I and II DNA and implications for DNA barcoding. Mol. Phylogenet. Evol. 44, 325–345. doi:10.1016/j.ympev.2006.12.005 Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–4.
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Salinas, I., Anderson, S.A., Wright, J., Webb, V.L., 2012. In vivo innate immune responses of groper (Polyprion oxygeneios) against Miamiensis avidus infection and lack of protection following dietary vitamin C administration. Fish Shellfish Immunol. 32, 8–15. doi:10.1016/j.fsi.2011.08.017
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Salinas, I., Maas, E.W., Muñoz, P., 2011. Characterization of acid phosphatases from marine scuticociliate parasites and their activation by host’s factors. Parasitology 138, 836–47. doi:10.1017/S0031182011000527
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Smith, P.J., McVeagh, S.M., Hulston, D., Anderson, S. A, Gublin, Y., 2009. DNA identification of ciliates associated with disease outbreaks in a New Zealand marine fish hatchery. Dis. Aquat. Organ. 86, 163–7. doi:10.3354/dao02113
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Song, J.Y., Kitamura, S.-I., Oh, M.-J., Kang, H.-S., Lee, J.-H., Tanaka, S.-J., Jung, S.-J., 2009. Pathogenicity of Miamiensis avidus (syn. Philasterides dicentrarchi), Pseudocohnilembus persalinus, Pseudocohnilembus hargisi and Uronema marinum (Ciliophora, Scuticociliatida). Dis. Aquat. Organ. 83, 133– 143. doi:10.3354/dao02017 Sterud, E., Hansen, M.K., Mo, T.A., 2000. Systemic infection with Uronema-like ciliates in farmed turbot, Scophthalmus maximus ( L .). J. Fish Dis. 23, 33–37. Tamura, K., Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725-2729. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL _ X windows interface : flexible strategies for multiple sequence alignment aided by quality analysis tools 25, 4876–4882. Whang, I., Kang, H., Lee, J., 2013. Identification of scuticociliates (Pseudocohnilembus persalinus, P. longisetus, Uronema marinum and
ACCEPTED MANUSCRIPT Miamiensis avidus) based on the cox1 sequence. Parasitol. Int. 62, 7–13. Whang, I., Kang, H.-S., Lee, J., 2011. Morphological and molecular characterization of Pseudocohnilembus longisetus Thompson, 1965 from farmed black rockfish Sebastes schlegelii in Korea. Vet. Parasitol. 179, 227– 33. doi:10.1016/j.vetpar.2011.02.009
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Young, N.D., Crosbie, P.B.B., Adams, M.B., Nowak, B.F., Morrison, R.N., 2007. Neoparamoeba perurans n. sp., an agent of amoebic gill disease of Atlantic salmon (Salmo salar). Int. J. Parasitol. 37, 1469–1481. doi:10.1016/j.ijpara.2007.04.018
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Table 2: Short sub-unit ribosomal DNA (SSU rDNA) scuticociliate sequences used in this study (not including those collected from SBT the present study)
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Table 3: Mitochondrial cytochrome C oxidase 1 (mt cox1) scuticociliate sequences used in analysis (not including those from present study)
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Figures
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Figure 1: Bayesian Inference Analysis of partial SSU rDNA of scuticociliates via Mr Bayes v 3.2.2. Tetrahymena pyriiformis was the outgroup.
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Figure 2: Bayesian Inference Analysis of partial mitochondrial cytochrome c oxidase 1 gene of scuticociliates via Mr Bayes v 3.2.2. Tetrahymena pyriiformis was the outgroup.
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Primer
Sequence
Target
OX-09-142
5’- AGTAATAATAGAACATTTAACGAATTTAATAACAC
M. avidus cox1
OX09-143
5’- CGTCTTGTAATTAATAAATTTGTAAACGATAC AACATAGAGCATATAGAGAGTACTCTAA
M. avidus cox1
OX09-144
5’- AACATAGAGCATATAGAGAGTACTCTAA
U. marinum cox1
OX09-145
5’- TTCATCCAGCTGTTGTTAATGT
U. marinum cox1
Cil 3
5’- GTAGGCTCTTTACCTTGA
SSU rRNA of scuticociliates
Cil 4
5’- CAAATCACTCCACCAACT
SSU rRNA of scuticociliates
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Source
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Cohnilembus verminus isolate FXP Entorhipidium tenue Homalogastra setosa isolate GT1 Mesanophrys carcini Metanophrys similis Metanophrys sinensis isolate FXP Miamiensis avidus 1 Miamiensis avidus 2 Miamiensis avidus isolate JM1 Miamiensis avidus isolate JM2 Miamiensis avidus strain A3 Miamiensis avidus strain GJ01 Miamiensis avidus strain JJ3 Miamiensis avidus strain JJ4 Miamiensis avidus strain SJF-03B Miamiensis avidus strain SJF-06A Miamiensis avidus strain WD4 Miamiensis avidus strain I1 Miamiensis avidus* strain SNUSS001 Miamiensis sp. 2 PJS-2009 Paranophrys magna Parauronema longum Philaster apodigitiformis Philasterides armatalis strain G Plagiopyliella pacifica Porpostoma notata isolate FXP Pseudocohnilembus hargisi Pseudocohnilembus longisetus Pseudocohnilembus persalinus 3 Pseudocohnilembus persalinus 1 Pseudocohnilembus persalinus 2 Pseudocohnilembus persalinus isolate wyg Schizocaryum dogieli Tetrahymena pyriformis (Outgroup) Thyrophylax vorax Uronema marinum 2 Uronema marinum 1 Uronema marinum strain CESP/1 Uronema marinum strain CLIT/I Uronema marinum strain CPAL2/I Uronema marinum strain CTAS/I Uronema marinum strain JK1 Uronema marinum strain JK2 Uronema marinum strain JK3 Uronema sp. 1 PJS-2009 Uronema sp. WS-2012 isolate XY2009113003 Uronemella filificum
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Schizocaryum dogieli Tetrahymena pyriformis Thyrophylax vorax Uromena marinum
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Miamiensis sp.. Paranophrys magna Parauronema longum Philaster apodigitiformis Philasterides armatalis Plagiopyliella pacifica Porpostoma notata Pseudocohnilembus hargisi P. longisetus P. persalinus
Uronema sp. Uronema sp. U. filificum
HM236339.1 AY541688.1 EF158848.1 AY103189.1 AY314803.1 HM236336.1 AY550080.1 AY642280.1 JN689229.1 JN689230.1 EU831193.1 EU831199.1 EU831194.1 EU831198.1 EU831195.1 EU831196.1 EU831192.1 JX914665.1 GU572375.1 FJ936000.1 AY103191.1 AY212807.1 FJ648350.1 FJ848877.1 AY541685.1 HM236335.1 AY833087.1 FJ899594.1 AY835669.1 AY551906.1 GU584096.1 GQ265955.1 AF527756.1 X56171.1 AY541686.1 Z22881.1 AY551905.1 GQ259744.1 GQ259745.1 GQ259746.1 GQ259747.1 DQ867072.1 DQ867073.1 DQ867074.1 FJ936001.1 JN885088.1 EF486866.1
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Cohnilembus verminus Entorhipidium tenue Homalogastra setosa Mesanophrys carcini Metanophrys similis Me. sinensis Miamiensis avidus
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Name in Phylogenetic tree Anophyroides haemophila
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Taxonomic name Anophyroides haemophila
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Entodiscus borealis Miamiensis avidus
Entodiscus borealis isolate OLI Miamiensis avidus 2 Miamiensis avidus isolate IUET-AK26P
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Pseudocohnilembus longisetus
Miamiensis avidus strain A3 Miamiensis avidus strain Iyo-1 Miamiensis avidus strain Mie0301 Miamiensis avidus strain Nakajima Miamiensis avidus strain SJF-03B Miamiensis avidus strain WD4 Miamiensis avidus strain YS3 Miamiensis avidus 1 Pseudocohnilembus longisetus
Tetrahymena pyriformis
Pseudocohnilembus persalinus 1 Pseudocohnilembus persalinus 2 Tetrahymena pyriformis strain E (Outgroup)
Uronema marinum
Uronema marinum
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P. persalinus
GenBank Accession no. FJ905123.1 GQ855300.1 KP170494.1 EU831214.1 EU831227.1 EU831233.1 EU831226.1 EU831216.1 EU831213.1 EU831218.1 GQ342957.1 GQ500580.1
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Name in Phylogenetic tree
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Taxonomic name
GQ500579.1 GU584095.1 EF070300.1 GQ500578.1
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Research highlights · Miamiensis avidus is associated with swimmer syndrome affected Southern bluefin tuna (SBT) Thunnus maccoyii ranched off Port Lincoln, Australia.
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· Molecular characterisation of Short sub-unit (SSU) rDNA and mitochondrial c oxidase 1 (cox1) of scuticociliates obtained from SBT affected by swimmer syndrome identified them as M. avidus.
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· Bayesian Inference analysis of SSU rDNA and cox1 showed that scuticociliate isolates from SBT forms a clade with M.
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avidus collected from other teleosts to the exclusion of other scuticociliates.
· First time M. avidus reported from Australian waters
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· Based on our findings we did not find any evidence of Uronema spp. being present in infected SBT
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Jimena Balli Garza, Nathan J. Bott, Michael D. Hammond, Natalie Shepherd, Barbara F. Nowak PII: DOI: Reference:
S0044-8486(16)30497-5 doi: 10.1016/j.aquaculture.2016.11.040 AQUA 632437
To appear in:
aquaculture
Received date: Revised date: Accepted date:
17 September 2016 21 November 2016 29 November 2016
Please cite this article as: Jimena Balli Garza, Nathan J. Bott, Michael D. Hammond, Natalie Shepherd, Barbara F. Nowak , Molecular characterisation of Miamiensis avidus (Ciliophora: Scuticociliata) from ranched Southern Bluefin tuna, Thunnus maccoyii off Port Lincoln, South Australia. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Aqua(2016), doi: 10.1016/ j.aquaculture.2016.11.040
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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1. Institute for Marine and Antarctic Studies, University of Tasmania, Locked Bag 1370, Launceston 7250, Tasmania, Australia 2. Centre for Environmental Sustainability and Remediation, School of Science, RMIT University, PO Box 71, Bundoora 3083, Victoria, Australia.
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*-Corresponding author: Nathan Bott, Centre for Environmental Sustainability and Remediation, School of Science, RMIT University, PO Box 71, Bundoora 3083, Victoria, Australia. Ph. +61 3 99257143, Fax +61 3 99257110, Email. [email protected]
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† equal first authors
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Abstract Scuticociliates are opportunistic protozoan pathogens present in a wide range of teleost hosts. Uronema spp. and Miamiensis spp. are the two most common genera recorded from scuticociliatosis cases in farmed and ornamental fish. Southern bluefin tuna (Thunnus maccoyii) (SBT) ranching is a high value aquaculture sector, situated off Port Lincoln, South Australia. Uronema nigricans has been previously associated with SBT swimmer mortality syndrome and was considered to be the causative agent. We conducted the first molecular characterisation of swimmer syndrome agent from affected SBT. Comparison of SSU rDNA and mitochondrial cytochrome c oxidase 1 sequences from the cerebrospinal fluid from SBT affected by swimmer syndrome and Uronema marinum samples, and phylogenetic analyses identified the scuticociliate present in SBT samples as Miamiensis avidus. Bayesian Inference analyses of both partial gene sequences of the Port Lincoln isolates form a clade with known M. avidus to the exclusion of Uronema spp. This shows that M. avidus is associated with swimmer syndrome and is present in the environment around SBT leases. Based on our molecular data, there is no evidence of Uronema spp. presence in the infected SBT. This is the first time M. avidus has been documented in Australia.
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Jimena Balli Garza1†, Nathan J. Bott2*†, Michael D. Hammond2, Natalie Shepherd2, Barbara F. Nowak1
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Molecular characterisation of Miamiensis avidus (Ciliophora: Scuticociliata) from ranched Southern Bluefin tuna, Thunnus maccoyii off Port Lincoln, South Australia
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Key words: Miamiensis avidus, Thunnus maccoyi, Scuticociliates, molecular characterisation, Southern bluefin tuna
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Introduction Scuticociliates are free-living marine organisms which feed on suspended bacteria, microalgae or other protozoa. Under certain circumstances scuticociliates
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can behave as opportunistic histophagous parasites of marine fish (Elston et al.,
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1999; Lee et al., 2004; Moustafa et al., 2010). They are considered to be serious
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pathogens in fish mariculture (Budiño et al., 2011; Gao et al., 2012, 2010; Whang
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et al., 2011) and were responsible for causing mortalities in olive flounder
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Paralichthys olivaceus (see Iglesias et al., 2001; Jung et al., 2005; Moustafa et al.,
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2010), turbot Scophthalmus maximus (see Dyková and Figueras, 1994; Iglesias et
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al., 2001; Sterud et al., 2000; Whang et al., 2013), sea bass Dicentrarchus labrax
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(see Whang et al., 2013), Southern bluefin tuna Thunnus maccoyii (SBT) (Munday
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et al., 1997, 2003), New Zealand grouper Polyprion oxygeneios and Yellowtail
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kingfish Seriola lalandi (Smith et al., 2009).
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The SBT industry is a high value aquaculture sector situated off Port
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Lincoln, South Australia. The industry practices purse seine fishing to collect 2-
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4year age class SBT in the Great Australian Bight, towing the fish back to the
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commercial lease sites and fattening over a 6 month period. Marine scuticociliates
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are the causative agent of swimmer syndrome in SBT, the disease typically occurs
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between May and November when water temperature drops below 18°C and, in
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most cases is associated with water temperature below 15°C (Deveney et al.,
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2005; Nowak et al., 2007). Clinical signs include abnormal and vigorous swimming
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at the surface followed by death (Munday et al., 1997). Affected fish are
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characterised by significant pathological changes in the olfactory rosettes and brain
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(encephalitis) along with the presence of scuticociliates. These ciliates have been
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identified as Uronema nigricans based on morphological analysis of cultures from
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cerebrospinal fluid (CSF) of infected SBT (Munday et al., 2003).
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In 2003, 58% of SBT mortalities examined from a mortality outbreak in
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winter were identified as being positive for scuticociliates based on the presence of
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scuticociliates in CSF (Deveney et al., 2005). This outbreak was affecting SBT
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from cages belonging to one company. Improved husbandry and feeding practices
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in the industry have decreased disease prevalence, however sporadic outbreaks
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still occur (Deveney et al., 2005; Nowak et al., 2007; Nowak, 2007).
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The use of morphological features to identify ciliates can be difficult (Jung et al., 2007; Song et al., 2009). Scuticociliates display considerable morphological
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plasticity, especially when cultured in vitro (Budiño et al., 2011; Salinas et al.,
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2012). Molecular techniques can be used to identify scuticociliates (Whang et al.,
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2013). Analysis of ribosomal RNA (rRNA) subunits has proven useful for
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identification and comparison of closely related organisms, including congeners
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(Elwood et al., 1985; Hillis and Dixon, 1991), while the application of mitochondrial
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barcoding of cytochrome c oxidase 1 (cox1) gene has been increasingly used for
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species delineation (Hebert et al., 2003; Robba et al., 2006; Roe and Sperling,
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2007). The use of sequencing data of rRNA genes and cox1 is a universally
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applicable tool that makes it possible to identify scuticociliates and confirm
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taxonomic relationships previously established on the basis of ultrastructural and
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other morphological characteristics (Budiño et al., 2011; Elwood et al., 1985; Hillis
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and Dixon, 1991; Jung et al., 2005; Whang et al., 2013).
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In this study we document the molecular characterisation of scuticociliates isolated from CSF of suspected swimmer syndrome SBT mortalities and record the
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first identification of the scuticociliate Miamiensis avidus (senior synonym of
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Philasterides dicentrachi (Budiño et al., 2011; Jung et al., 2007)) from SBT (new
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host) in Australia (new location). We provide data that the scuticociliates isolated
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from SBT are distinct from the previously reported agent of swimmer syndrome
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Uronema nigricans.
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Materials and methods
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Field collection and processing of Southern Bluefin Tuna
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During 2015, 16 wild SBT caught from Pedra Branca off the Southern coast of Tasmania (45o51’00”S 146o58’12”E), were collected by long line fishing.
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Individual olfactory rosettes were removed from the 16 SBT. In addition, 23
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cerebrospinal fluid (CSF) samples (olfactory rosettes not available) were collected
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from ranched SBT which exhibited signs of swimmer syndrome, such as vigorous
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swimming inside the cage or at intervals swimming up to the surface and sinking
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again. SBT were opportunistically sampled and ranged between 15-40kg. CSF
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was sampled from fresh mortalities and moribund individuals using plastic Pasteur
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pipettes during the 2015 harvesting season from May to the second week of
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August, off Port Lincoln, South Australia (34o43’56”S 135o51’31”E). Prior to storage
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at -20oC, CSF subsamples were examined for the presence of live scuticociliates.
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Wet preparations of subsamples were examined and observed using a bright-field
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microscope at 100X and 400X and most were found to be positive. Samples were
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considered positive for scuticociliates if the microscopic observation revealed
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motile cells that were robust and granular in appearance (Jung et al., 2007) and
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pyriform shape (morphology consistent for scuticociliates). All samples (olfactory
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rosettes and CSF) were stored in nucleic acid preservation solution (4 M
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ammonium sulphate, 25 mM sodium citrate, 10 mM EDTA; pH 5.5) in a 1:5 ratio.
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All samples were stored on ice at approximately 4oC overnight and then at -20°C in
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the laboratory.
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Nucleic acid extraction and molecular analysis Total nucleic acid (TNA) was extracted from the RNA-later preserved olfactory rosettes and CSF samples using Bioline Isolate II Genomic DNA Kit
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(Bioline, Taunton, MA, USA), following the manufacturer’s instructions. TNA
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quantity of each sample was estimated using spectrophotometry (NanoDrop,
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Thermo Scientific, Waltham MA, USA). Additionally, DNA samples, courtesy of
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Prof. Iva Dyková (Brno University, Czech Republic), from cultured scuticociliates
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(Dyková et al., 2010) were provided; two of which were isolated in conjunction with
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Neoparamoeba perurans from fish hosts Psetta maxima (CESP/I) and Salmo salar
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(CTAS/I), and two from seaweeds Lithophyllum racemus (CLIT/I) and Palmeria
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palmata (CPAL2/1). These samples were also used for the molecular analysis and
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sequence comparison to scuticociliates from SBT. Partial fragments of the
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mitochondrial cytochrome c oxidase subunit 1 (cox1) gene (360 nucleotides) and
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the small subunit rDNA (SSU rDNA) (507 nucleotides) sequence were amplified
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using oligonucleotide primers previously reported by Jung et al., 2005 and Whang
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et al., (2013) (Table 1). PCR was performed in a final volume of 25 μl containing, 10 pM of each
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primer, 0.5 U of DNA Taq Polymerase (Bioline, Taunton, MA USA), 1.5 mM MgCl2,
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0.5 mM dNTPs, stabilizers and enhancers (MyTaq, Bioline, Taunton, MA USA),
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and DNA template. PCR amplification was performed in a Bio-Rad C1000 Touch
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Thermal Cycler (Bio-Rad, Hercules, CA USA). The following conditions were used
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to amplify partial SSU rDNA fragment, using primers Cil 2/Cil 4: at 95oC for 1 min
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(activation) and 40 cycles of 95oC for 15 s (denaturing), 50oC for 15 s (annealing),
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72oC for 15s (extension) and a final hold at 4oC. The PCR conditions used to
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amplify a partial SSU rDNA fragment using Cil 3/Cil 4 were 95oC for 1 min
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(activation) and 35 cycles of 95oC for 15 s (denaturing), 50oC for 15 s (annealing),
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72oC for 15s (extension) and 4oC as a final hold. The PCR conditions used to
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amplify a partial sequence of the cox1 gene of M. avidus using primers OX09-142,
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OX09-143 and a partial fragment of the cox1 gene of U. marinum using OX09-144,
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OX09-145 were: 95oC for 1 min (activation) and 30 cycles of 95oC for 15 s
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(denaturation), 50oC for 15 s (annealing) 72oC for 15 s (extension) and 4oC as a
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final hold.
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Amplified products were analysed in a 1.5% agarose gel by electrophoresis,
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stained with ethidium bromide and visualized using the UV transilluminator Gel Doc
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XR+ System (Bio-Rad, Hercules, CA USA). Amplified products were purified using
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the ISOLATE II PCR and Gel Purification kit (Bioline, Taunton, MA USA) and
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concentration was measured by spectrophotometry (NanoDrop, Thermo Scientific,
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Waltham MA, USA). Both strands, forward and reverse of the PCR products, were
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sequenced using ABI Big Dye Terminator v3.1 chemistry (Applied Biosystems,
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Foster City, CA USA) by the Australian Genome Reference Facility (AGRF),
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Parkville, Victoria.
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Alignment and Phylogenetic analysis Sequencher™ (GeneCodes Corp., Ann Arbor, Michigan, U.S.A, ver. 5.2.4) was used to produce consensus sequences from corresponding forward and
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reverse complemented sequences. Scuticociliate sequences from the appropriate
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gene regions (partial SSU rDNA and mt cox 1) were obtained via BLAST search
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(Altschul et al., 1990) (Tables 2 and 3) and aligned in conjunction with the
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sequences obtained as part of this study using the Clustal X (Thompson et al.,
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1997) accessory application in Bioedit® (Hall, 1999). Alignments were further
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refined by eye. Sequences produced as part of this study are listed by locality (Port
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Lincoln) and numbered based on their sample number. Bayesian Inference
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analyses of sequence alignments were conducted using; MrBayes® ver. 3.2.2
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(Ronquist and Huelsenbeck, 2003) using the parameters: ngen=2,000,000, nst =
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six, four Markov Chains used, burn-in was set to 100 and every 100th tree saved.
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The trees were based on a 50% majority rule consensus as per Aiken et al.,
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(2007). The trees produced from Bayesian analyses were viewed using Figtree ®
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(http://tree.bio.ed.ac.uk/software/figtree/). Multiple pairwise alignments were
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produced in Mega version 6 (Tamura et al. 2013) using number of differences.
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Partial SSU rDNA and mt Cox 1 sequences generated from this study were
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deposited in Genbank under the accession numbers KX842459-KX842468 (M.
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avidus SSU rDNA), KX842469-KX842477 (M. avidus mt Cox 1) and KX842478-
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KX842483 (U. marinum mt Cox 1).
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Results
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Molecular identification of scuticociliates Nineteen samples from CSF of ranched SBT were identified as positive for scuticociliates using PCR. None the olfactory rosettes samples from wild SBT
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considered negative. The scuticociliates isolated from CSF of SBT were 100%
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identical based on both partial SSU rDNA and mt cox1. Both partial SSU rDNA and
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mt cox1 showed that the scuticociliates isolated from SBT CSF were identical to
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published sequences of M. avidus and differed from Uronema spp. sequenced as
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part of this study and previously published sequences; partial SSU rDNA differed
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by 23 - 24 nucleotides and 95% similarity, and mt cox1 differs by a range of 34-35
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nucleotides and is 87.5-90% in range of similarity. Our sequencing results for SSU
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rDNA of U. marinum isolates from Dykova et al. (2010) showed that their
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sequences were 100% identical to those generated by Dyková et al. (2010) from
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the same samples. All U. marinum SSU rDNA sequences obtained in this study
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were 100% identical.
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Bayesian inference analyses of partial SSU rDNA (Figure 1) showed that the ten sequences from SBT ranched at Port Lincoln were 100% identical and
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homologous to published SSU rDNA sequences of M. avidus (Jung et al., 2011,
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2007; Paramá et al., 2006; Salinas et al., 2012). The SBT samples and M. avidus
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formed a clade with all other taxa (to the exclusion of the Uronema clade including
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those sequenced as part of this study (samples from Dyková et al., 2010)).
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The partial mt cox1 Bayesian Inference analysis (Figure 2) showed a similar
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topology to the SSU tree in that M. avidus formed a clade with Pseudocohnilembus
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spp. to the exclusion of the Uronema + Entodiscus borealis clade. Within the M.
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avidus + Pseudocohnilembus spp. clade, M. avidus formed a clade to the
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exclusion of Pseudocohnilembus spp.; the samples collected from Pt Lincoln as
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part of this study formed a clade to the exclusion of all other M. avidus.
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Discussion
The comparison of SSU rDNA and mt cox1 sequences of scuticociliates collected from SBT and U. marinum in association with comparison to published
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M. avidus was present and U. nigricans which was identified during earlier SBT
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ranching seasons as a cause of swimmer syndrome (Crosbie and Munday, 1997;
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Deveney et al., 2005; Munday et al., 1997, 2003), was absent. Previous
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identification was based on morphology of cultures of the ciliates from SBT CSF
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(Munday et al., 1997) and it has been shown that morphology of scuticocilliates
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can change in culture (Fenchel, 1990; Jee et al., 2001). Our phylogenetic analyses
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of 2 gene regions (SSU rDNA and mt cox1) (Figure 1 and 2) that are subjected to
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different evolutionary pressures (Gao et al., 2012; Jung et al., 2011a) clearly
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showed that the samples isolated from SBT swimmer syndrome samples were M.
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avidus, and based on these analyses they were not particularly close in sequence
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similarity to the Uronema sp. sequenced in this study.
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Our Bayesian Inference analyses support the work of Gao et al., (2012) in illustrating that the genera belonging to Philasterida: Miamiensis and Uronema are
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consistently positioned within different clades. Our study differed from Gao et al.,
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(2012) in that we have analysed only within the Philasterida while they compared
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SSU rDNA of a wide range of orders within the Scuticociliata. The partial SSU
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rDNA analysis positioned the Uronema clade to the exclusion of all other
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scuticociliates as opposed to that of the complete SSU rDNA in Gao et al., (2012);
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this may be an artefact of not analysing the complete gene region.
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While our results show that M. avidus is associated with swimmer syndrome
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in SBT further investigation is required to determine if Uronema nigricans (or
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indeed other opportunistic scuticociliate species) is also associated with swimmer
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syndrome in ranched SBT. If multiple species are causative agents and U.
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nigricans is also associated with swimmer syndrome in SBT (as described by
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Munday et al., 1997) then an investigation is warranted into whether environmental
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cues favour infection by one scuticociliate species over another. It is plausible that
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both scuticociliate species (M. avidus and U. nigricans) are responsible as they are
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Mixed isolates of scuticocilliates, including M.avidus, U. marinum and
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Parauronema virginianum were obtained from infected New Zealand grouper,
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Polyprion oxygeneios (see Salinas et al., 2011). Smith et al. (2009) isolated from
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dead P. oxygeneios and identified using SSU rRNA both M. avidus and U.
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marinum. However, in some host species only M. avidus has been suggested as
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the cause of scuticociliatosis. Only M. avidus induced histological changes when
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olive flounder Paralichthys olivaceus were experimentally infected with M. avidus,
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Pseudocohnilembus persalinus, P. hargisi and U. marinum. (Song et al., 2009).
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This suggested that P. persalinus, P. hargisi and U. marinum were not causative
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agents of scuticociliatiosis in olive flounder, although all three species have been
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reported and identified in the affected fish (Song et al 2009) and Smith et al. (2009)
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reported that U. marinum caused mortalities in Seriola lalandi in New Zealand.
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Culture conditions may promote growth of one species, and in some cases that
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species may not be the pathogen. For example, cultures of amoebae from the gills
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of Amoebic Gill Disease-affected Atlantic salmon resulted in isolation of N.
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pemaquidensis and N. branchiphila, while it was later shown that N. perurans is the
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only causative agent (Crosbie et al., 2012; Young et al., 2007). Further
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investigation is required to assess the broad pathogenicity of a range of
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scuticociliates believed to cause scuticociliatosis in finfish. This study is the first to
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document the presence of M. avidus in Australia. It is yet to be established how
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widespread M. avidus is in Australia’s marine environment.
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Acknowledgements
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The authors would like to thank the Australian Southern Bluefin Tuna Industry
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Association for their ongoing support, in particular Kirsten Rough and Claire
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Webber. We would also like to thank Dr Daryl Evans, Marnikol Fisheries for his
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ongoing support of SBT Health research. We are grateful to Prof. Iva Dyková for
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providing the DNA samples of Uronema spp. All work with animals and methods
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for recovering samples were approved by the University of Tasmania Animal Ethics
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Committee, project number A0013175. Samples from Czech Republic were
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imported under Quarantine permit IP15004906.
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Song, J.Y., Kitamura, S.-I., Oh, M.-J., Kang, H.-S., Lee, J.-H., Tanaka, S.-J., Jung, S.-J., 2009. Pathogenicity of Miamiensis avidus (syn. Philasterides dicentrarchi), Pseudocohnilembus persalinus, Pseudocohnilembus hargisi and Uronema marinum (Ciliophora, Scuticociliatida). Dis. Aquat. Organ. 83, 133– 143. doi:10.3354/dao02017 Sterud, E., Hansen, M.K., Mo, T.A., 2000. Systemic infection with Uronema-like ciliates in farmed turbot, Scophthalmus maximus ( L .). J. Fish Dis. 23, 33–37. Tamura, K., Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725-2729. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL _ X windows interface : flexible strategies for multiple sequence alignment aided by quality analysis tools 25, 4876–4882. Whang, I., Kang, H., Lee, J., 2013. Identification of scuticociliates (Pseudocohnilembus persalinus, P. longisetus, Uronema marinum and
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Young, N.D., Crosbie, P.B.B., Adams, M.B., Nowak, B.F., Morrison, R.N., 2007. Neoparamoeba perurans n. sp., an agent of amoebic gill disease of Atlantic salmon (Salmo salar). Int. J. Parasitol. 37, 1469–1481. doi:10.1016/j.ijpara.2007.04.018
ACCEPTED MANUSCRIPT Tables Table 1. Oligonucleotides used in polymerase chain reactions for this study.
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Table 2: Short sub-unit ribosomal DNA (SSU rDNA) scuticociliate sequences used in this study (not including those collected from SBT the present study)
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Table 3: Mitochondrial cytochrome C oxidase 1 (mt cox1) scuticociliate sequences used in analysis (not including those from present study)
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Figure 1: Bayesian Inference Analysis of partial SSU rDNA of scuticociliates via Mr Bayes v 3.2.2. Tetrahymena pyriiformis was the outgroup.
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Figure 2: Bayesian Inference Analysis of partial mitochondrial cytochrome c oxidase 1 gene of scuticociliates via Mr Bayes v 3.2.2. Tetrahymena pyriiformis was the outgroup.
ACCEPTED MANUSCRIPT Table 1
Primer
Sequence
Target
OX-09-142
5’- AGTAATAATAGAACATTTAACGAATTTAATAACAC
M. avidus cox1
OX09-143
5’- CGTCTTGTAATTAATAAATTTGTAAACGATAC AACATAGAGCATATAGAGAGTACTCTAA
M. avidus cox1
OX09-144
5’- AACATAGAGCATATAGAGAGTACTCTAA
U. marinum cox1
OX09-145
5’- TTCATCCAGCTGTTGTTAATGT
U. marinum cox1
Cil 3
5’- GTAGGCTCTTTACCTTGA
SSU rRNA of scuticociliates
Cil 4
5’- CAAATCACTCCACCAACT
SSU rRNA of scuticociliates
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Whang et al. 2013
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Source
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ACCEPTED MANUSCRIPT Table 2 GenBank Accession no. U51554.1
Cohnilembus verminus isolate FXP Entorhipidium tenue Homalogastra setosa isolate GT1 Mesanophrys carcini Metanophrys similis Metanophrys sinensis isolate FXP Miamiensis avidus 1 Miamiensis avidus 2 Miamiensis avidus isolate JM1 Miamiensis avidus isolate JM2 Miamiensis avidus strain A3 Miamiensis avidus strain GJ01 Miamiensis avidus strain JJ3 Miamiensis avidus strain JJ4 Miamiensis avidus strain SJF-03B Miamiensis avidus strain SJF-06A Miamiensis avidus strain WD4 Miamiensis avidus strain I1 Miamiensis avidus* strain SNUSS001 Miamiensis sp. 2 PJS-2009 Paranophrys magna Parauronema longum Philaster apodigitiformis Philasterides armatalis strain G Plagiopyliella pacifica Porpostoma notata isolate FXP Pseudocohnilembus hargisi Pseudocohnilembus longisetus Pseudocohnilembus persalinus 3 Pseudocohnilembus persalinus 1 Pseudocohnilembus persalinus 2 Pseudocohnilembus persalinus isolate wyg Schizocaryum dogieli Tetrahymena pyriformis (Outgroup) Thyrophylax vorax Uronema marinum 2 Uronema marinum 1 Uronema marinum strain CESP/1 Uronema marinum strain CLIT/I Uronema marinum strain CPAL2/I Uronema marinum strain CTAS/I Uronema marinum strain JK1 Uronema marinum strain JK2 Uronema marinum strain JK3 Uronema sp. 1 PJS-2009 Uronema sp. WS-2012 isolate XY2009113003 Uronemella filificum
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Schizocaryum dogieli Tetrahymena pyriformis Thyrophylax vorax Uromena marinum
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Miamiensis sp.. Paranophrys magna Parauronema longum Philaster apodigitiformis Philasterides armatalis Plagiopyliella pacifica Porpostoma notata Pseudocohnilembus hargisi P. longisetus P. persalinus
Uronema sp. Uronema sp. U. filificum
HM236339.1 AY541688.1 EF158848.1 AY103189.1 AY314803.1 HM236336.1 AY550080.1 AY642280.1 JN689229.1 JN689230.1 EU831193.1 EU831199.1 EU831194.1 EU831198.1 EU831195.1 EU831196.1 EU831192.1 JX914665.1 GU572375.1 FJ936000.1 AY103191.1 AY212807.1 FJ648350.1 FJ848877.1 AY541685.1 HM236335.1 AY833087.1 FJ899594.1 AY835669.1 AY551906.1 GU584096.1 GQ265955.1 AF527756.1 X56171.1 AY541686.1 Z22881.1 AY551905.1 GQ259744.1 GQ259745.1 GQ259746.1 GQ259747.1 DQ867072.1 DQ867073.1 DQ867074.1 FJ936001.1 JN885088.1 EF486866.1
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Cohnilembus verminus Entorhipidium tenue Homalogastra setosa Mesanophrys carcini Metanophrys similis Me. sinensis Miamiensis avidus
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Name in Phylogenetic tree Anophyroides haemophila
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Taxonomic name Anophyroides haemophila
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Entodiscus borealis Miamiensis avidus
Entodiscus borealis isolate OLI Miamiensis avidus 2 Miamiensis avidus isolate IUET-AK26P
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Pseudocohnilembus longisetus
Miamiensis avidus strain A3 Miamiensis avidus strain Iyo-1 Miamiensis avidus strain Mie0301 Miamiensis avidus strain Nakajima Miamiensis avidus strain SJF-03B Miamiensis avidus strain WD4 Miamiensis avidus strain YS3 Miamiensis avidus 1 Pseudocohnilembus longisetus
Tetrahymena pyriformis
Pseudocohnilembus persalinus 1 Pseudocohnilembus persalinus 2 Tetrahymena pyriformis strain E (Outgroup)
Uronema marinum
Uronema marinum
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P. persalinus
GenBank Accession no. FJ905123.1 GQ855300.1 KP170494.1 EU831214.1 EU831227.1 EU831233.1 EU831226.1 EU831216.1 EU831213.1 EU831218.1 GQ342957.1 GQ500580.1
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Name in Phylogenetic tree
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Taxonomic name
GQ500579.1 GU584095.1 EF070300.1 GQ500578.1
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Research highlights · Miamiensis avidus is associated with swimmer syndrome affected Southern bluefin tuna (SBT) Thunnus maccoyii ranched off Port Lincoln, Australia.
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· Molecular characterisation of Short sub-unit (SSU) rDNA and mitochondrial c oxidase 1 (cox1) of scuticociliates obtained from SBT affected by swimmer syndrome identified them as M. avidus.
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· Bayesian Inference analysis of SSU rDNA and cox1 showed that scuticociliate isolates from SBT forms a clade with M.
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avidus collected from other teleosts to the exclusion of other scuticociliates.
· First time M. avidus reported from Australian waters
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· Based on our findings we did not find any evidence of Uronema spp. being present in infected SBT
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